The objective of this project is to study at the molecular level the mechanism by which the genes responsible for the phosphate global metabolism exert their function. In E. coli, 7-10 regulatory genes interact in a cascade fashion to control the expression of 15-20 structural genes (the Pho Regulon). One of these is the gene phoA of alkaline phosphatase, whose expression will be monitored. The project entails the reconstitution in vitro of the interacting regulatory gene products. It requires: 1) genetic manipulations to obtain efficient multicopy plasmids of each gene in order to overproduce and isolate the proteins; 2) preparation of specific DNA templates from plasmids carrying phoA and the regulatory genes (phoB, phoR, phoM); 3) isolation of specific nucleotide cofactors identified as positive and negative effectors. In vivo it will be possible to analyze the physiological effect of various levels of regulatory products by preparing gene fusions (like tacp-phoB) whose promoter will be regulated by the concentration of the inducer (in this case, IPTG). The genetic approach used will also allow the isolation of novel mutants of phoB and phoR.